The Toxoplasma micropore mediates endocytosis for selective nutrient salvage from host cell compartments

Apicomplexan parasite growth and replication relies on nutrient acquisition from host cells, in which intracellular multiplication occurs, yet the mechanisms that underlie the nutrient salvage remain elusive. Numerous ultrastructural studies have documented a plasma membrane invagination with a dense neck, termed the micropore, on the surface of intracellular parasites. However, the function of this structure remains unknown. Here we validate the micropore as an essential organelle for endocytosis of nutrients from the host cell cytosol and Golgi in the model apicomplexan Toxoplasma gondii. Detailed analyses demonstrated that Kelch13 is localized at the dense neck of the organelle and functions as a protein hub at the micropore for endocytic uptake. Intriguingly, maximal activity of the micropore requires the ceramide de novo synthesis pathway in the parasite. Thus, this study provides insights into the machinery underlying acquisition of host cell-derived nutrients by apicomplexan parasites that are otherwise sequestered from host cell compartments.


Supplementary Figures 1-12 and Legends
Supplementary Fig. 1. Discovery of proteins that are likely to be associated with endocytosis in T. gondii.
a Table list of proteins likely to be associated with endocytosis in T. gondii.
b Proteins listed in (a) were localized to discrete spots on the plasma membrane or to other structures, showing IFA results with Ty-tagged proteins (green) and GAP45 (red) as the control. Scale = 5 µm.
c IFA and western blot detection of TurboID labeling by the fusion of EPS15.
Biotinylated proteins were detected using Streptavidin Alexa Fluor-488 (Strep-488) in the parental and TurboID fusion parasites grown for 24 hr and treated with 500 µM biotin for 1 hour. The Strep-488 signals (green) were co-localized with Ty staining (red) at the pellicular foci and additional green foci were the signals from the apicoplast. Western blot detection using streptavidin LICOR 800CW were performed for the parasites grown and treated with biotin for different times as indicated. ALD, aldolase as a control. Scale = 5 µm.
d Volcano plot analysis comparing the TurboID fusion to the control line RH (fold change > 2) using replicate mass-spectrometry datasets. Hits were analyzed by twosided student t test, and significant hits were shown in numbers and indicated by colors. Detailed information on the hits were listed in Supplementary Table 4.  Table 4) were endogenously tagged with 6Ty for IFA analysis using Ty antibodies (green) and IMC1 (red), followed by secondary antibodies conjugated with Alexa Fluor reagents. Five new proteins were localized at the discrete spots similar to the bait EPS15, as observed in (b). b, c, e, three independent experiments were performed for IFA and western blots with similar outcomes, and representative images are shown. c Intrinsic disordered regions were predicted by a newly developed webserver fIDPnn 1 . This webserver predicts the intrinsic disordered regions together with the possible protein binding, DNA binding and RNA binding. Protein binding were found for proteins of K13, AP2αn, MPP1, UBP1, PPG1 and AP2αc. (green) were co-localized with the Ty fusion (red), and the additional green spots were the apicoplast. The parasites were grown in D5 for 24 hours, and treated with 500 µM biotin for 1 hour before fixation for IFA. AP2µ-TurboID was used as a representative for the AP2 components, as other TurboID fusions were not generated successfully. GAP45 was stained green as a control for the IFA. Scale = 5 µm.

c-d Diagnostic PCR of the AID lines. The endogenous (endo) and integration (integ)
PCRs were performed using primers illustrated in (a-b), using a general TAQ DNA polymerase with a PCR extension time of 1 minute. DNA used for the PCR was extracted from the TIR1 and AID lines, and the promoter region of tubulin was used as the PCR control (contr). Three independent experiments were performed with similar outcomes.
The endo PCR detected the specific endogenous DNA in the genome in the TIR1 line, but not in the AID lines, while the integ PCR detected the gene fusion in the AID lines but not in the TIR1 line. The diagnostic PCR were performed for three genes (i.e. EPS15, K13 and PPG1) in the lines of dKD and tKD. The stars (*) indicated the nonspecific bands for the endoPCR products. We observed that the endoPCR products for EPS15-AID (3' tagging) in the single AID (EPS15-AID), dKD and tKD appeared to be different. It has to be noted that the EPS15-AID line served as the parental line for the generation of the double AID (dKD) line and the triple AID (tKD) line. In addition, the nonspecific bands in the endoPCR for AID-PPG1 (5' tagging) in the single AID line (AID-PPG1) and the tKD line looked different as well. Compared to the PCR with the TIR1 samples that contain specific priming sequences, the endoPCR in the AID samples seemed to readily produce non-specific products and even differently produced non-specific products using different amounts of DNA in the sample. This is likely the reason for why we generally see more than one non-specific band. The diagnostic PCR clearly detected the specific PCR products (integ PCR) from the AID integration in the AID parasites, but not in the parental line TIR1. We thus conclude that the AID lines were clean clones that contained a specific integration of AID at the targeted genes of interest used in this study.
In addition, the primers for the endoPCR could still amplify a long PCR product that contains the integrated heterologous DNA fragment (the AID-resistant cassettes with sizes > 4.0 kbp). However, the reaction was performed with a general TAQ DNA polymerase in a short extension time. This won't allow the PCR to make the long PCR products, but might produce different lengths of DNA products. This would thus produce complex fragments of DNA in different reactions. Supplementary Fig. 4. IFA verification of all the AID lines used in the study.
Parasites were grown in ±IAA for 24 hr, followed by IFA analyses using antibodies against either Ty, HA or myc epitope fused with the AID degron. IMC1 served as a control for IFA. Scale = 5 µm. Three independent experiments were performed with similar outcomes, and one representative for each line was shown. Three independent experiments were performed with similar outcomes, and representatives of the images were shown. DIC, differential interference contrast. Scale = 5 μM. Supplementary Fig. 8. Parasites depleted with the micropore proteins were viable but lost fitness in the extracellular environment.
a Parasite egress was delayed in the lines grown in auxin for 36 hours. Parasites were stimulated by 3 μM A23187 for 5 min and 10 min, followed by IFA stains for GRA7 and IMC1 and by scorings of egressed and non-egressed vacuoles. The rates of egressed vacuoles at 10 min significantly increased compared to the rates at 5 min. Two independent experiments with triplicates were performed, and data are shown as a mean ± SD of the independent experiments with triplicates. At least 100 vacuoles were scored in each replicate.

b-c Extracellular parasites eventually lost fitness in protein-depleted parasites.
Parasites were grown in HFF in ±IAA for 36 hours prior to mechanical egress for analysis of invasion capabilities. Freshly egressed parasites (b) and these parasites incubated in DMEM for 3 hours (c) were used to challenge HFF monolayers for 30 min at 37°C. The invaded and non-invaded parasites were scored (n>100 parasites in each replicate) and plotted as invaded parasites per host cell nuclei. Two independent experiments with triplicates were performed for the lines. b Mitochondrial membrane potential detected by mitotracker red was lost in AID-K13 when grown in D5 media and in media containing glucose, but recovered when grown in media containing glutamine, after induction in auxin for 30 hours. The intracellular parasites were grown in the media containing different carbon sources as described above. The parasites were then treated with 500 nM mitotracker red CMXRos (ThermoFisher, M7512) in DMEM for 30 min, followed by fixation, permeabilization and staining with rabbit antibodies against hsp60 and secondary antibodies conjugated with Alexa Fluor-488. Two independent experiments were performed with similar outcomes. Scale = 5 μm. quantification outcomes were observed for the micropore defects and the PPM invagination close to the apex.